Molecular Mechanism Of Drug Metabolism In Shuanghuanglian

SHL composed by Honeysuckble,Baical Skullcap Root and Forsythia Suspensa,is a kind of compound preparation of Traditional Chinese Medicine,its main components are chlorogenic acid,bacalin and phillyrin.It mainly treats with diseases caused by affection of exogenous wind-heat such as fever,cough and malay kinds of upper respiratory infection. In this study,the liver protection function of SHL and the molecular mechanism of interaction between its three components were investigated by in vitro and in vivo experiments.In vitro study,rat primary cultured hepatocytes were isolated by two-step collagenase digestion method and treated with gradient concentration of SHL,chlorogenic acid,bacalin and phillyrin for 24h～72h respectively,and the expression of cytochrome P450 3A1 in hepatocytes was determined by Westem blot.The results as follows,(2～4)×10~8 cells per rat were obtained and the viability was about 95%.Hepatocytes grew well,had a good morphology and could be used for investigation of drug metabolism.CYP3A1 in rat hepatocytes had no induction with chlorogenic acid in 24h,but its expression increased gradually as the drug concentration increased in 48h and 72h.Treated with phillyrin for 24h～72h,the expression of CYP3A1 increased gradually as the drug concentration increased and time course.When the hepatocytes were treated with 20μM phillyrin for 72h, the induction to CYP3A1 was the most significant.Treated with bacalin for 24h～72h,the expression of CYP3A1 increased gradually as the drug concentration increased,and 10μM bacalin had the most significant induction.Treated with combinations of baicalin and chlorogenic acid,phillyrin and chlorogenic acid,phillyin and baicalin respectively for 24h～48h,the expression of CYP3A1 was positively correlated to the drug concentration and time.Treated with SHL for 24h～48h,the expression of CYP3A1 increased gradually as the drug concentration increased.Under the same concentration,the expression of CYP3A1 became more and more significant with time course.100μM SHL was the best concentration to induce CYP3A1.In vivo study,fifty SD rats were randomly divided into five groups(ten rats per group): Normal saline in control group,and different doses of SHL(100mg/kg,200mg/kg, 500mg/kg and 1000mg/kg)in four SHL groups were administrated.The drugs were administrated by intraperitoneal injection once a day,seven days later,all rats were put to death,the level of AST,ALT,LDH and GSH in the sera and the content of MDA,SOD in the liver homogenate were measured.The pathological changes of livers were detected by HE staining.The results as follows:All of the biochemical indicators and histopathology in livers were normal.Another forty SD rats were randomly divided into four groups(ten rats per group):Normal saline was given in control group and model group,Diammonium Glycyrrhizinate was given in positive drug group,and 200mg/kg SHL was given in SHL group,the drugs were admistrated by intraperitoneal injection once a day,on the seventh day,8h after the administration,300mg/kg APAP was injected into abdomen,the same volume of normal saline was given in the control group,kept without food but water.16h later,all rats were put to death,the level of ALT,AST,TP in the sera were measured,and pathological change of livers were detected by HE staining.The results as follows,the level of ALT and AST decreased significantly and the content of TP increased significantly in 200mg/kg SHL group(P＜0.01).The color of liver in model group became lighter and brittler,the surfaces of livers were coarse and the brittle particles were seen.The normal structure in the liver lobules destroyed,hepatic cords were disorder and decolled.The fat denaturation of cells was severity and the inflammation cells have been infiltrated and showed congestion.Compared with model group,the pathological changes of livers in SHL group alleviated obviously,no more fat denaturation was found,but congestion still be seen and seldom infiltration of inflammation cells.According to above results,we can obtain the conclusions that:CYP3A1 can be induced by SHL,chlorogenic acid,bacalin and phillyrin.The induction of CYP3A1 can be enhanced by combinations of baicalin and chlorogenic acid,phillyrin and chlorogenic acid, phillyin and baicalin.SHL has less toxicity to liver,can relieve the acute liver injury induced by APAP and has definite protective effects on liver injury.